Targeting non-coding RNAs with the CRISPR/Cas9 system in human cell lines.

Tsui-Ting Ho,Yin-Yuan Mo,Jianguo Huang,Min Xu,Roland Fung,Nanjiang Zhou,Pratirodh Koirala,Fangting Wu

doi:10.1093/nar/gku1198

Abstract

The CRISPR/Cas has been recently shown to be a powerful genome-editing tool in a variety of organisms. However, these studies are mainly focused on protein-coding genes. The present study aims to determine whether this technology can be applied to non-coding genes. One of the challenges for knockout of non-coding genes is that a small deletion or insertion generated by the standard CRISPR/Cas system may not necessarily lead to functional loss of a given non-coding gene because of lacking an open reading frame, especially in polyploidy human cell lines. To overcome this challenge, we adopt a selection system that allows for marker genes to integrate into the genome through homologous recombination (HR). Moreover, we construct a dual guide RNA vector that can make two cuts simultaneously at designated sites such that a large fragment can be deleted. With these approaches, we are able to successfully generate knockouts for miR-21, miR-29a, lncRNA-21A, UCA1 and AK023948 in various human cell lines. Finally, we show that the HR-mediated targeting efficiency can be further improved by suppression of the non-homologous end joining pathway. Together, these results demonstrate the feasibility of knockout for non-coding genes by the CRISPR/Cas system in human cell lines.

Highlights

It is well known that the human genome is actively transcribed; there are only about 20, 000 proteincoding genes [1], accounting for about 2% of the genome, and the rest of the transcripts are non-coding RNAs including microRNAs and long non-coding RNAs
A most commonly used approach for gene functional study is knockdown by RNA inference (RNAi) which is mainly functional in the cytoplasm where RISC complexes are located [3]
Type II restriction enzyme FokI is often used as a cleavage domain in zinc finger nuclease (ZFN) [7]; engineered TAL effectors can be fused to the cleavage domain of FokI to create transcription activation-like element nuclease (TALEN) for genome editing [8]

Summary

Introduction

It is well known that the human genome is actively transcribed; there are only about 20, 000 proteincoding genes [1], accounting for about 2% of the genome, and the rest of the transcripts are non-coding RNAs including microRNAs and long non-coding RNAs (lncRNAs). A novel genetic engineering tool called clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is more advanced because of easy generation and high efficiency of gene targeting [9]. It only requires changing the sequence of the guide RNA (gRNA); and it can be directly delivered into embryos, to generate sequence-modified animals [10,11,12,13]. Multiplexing capability of CRISPR/Cas makes it possible to target multiple genes simultaneously [12]

Methods

Results

Conclusion